Process for producing chloramphenicol analogs

ABSTRACT

A process for producing chloramphenicol analogs, which comprises culturing a bacterium capable of producing chloramphenicol analogs, belonging to the genus Corynebacterium or Nocardia and having a resistance to chloramphenicol or its analogs in a nutrient medium, and recovering chloramphenicol analogs from the culture liquor.

United States Patent [191 Suzuki et a1.

[ Nov. 12, 1974 PROCESS FOR PRODUCING CHLORAMPHENICOL ANALOGS .[75]Inventors: Takeo Suzuki; Fusao Tomita;

Hirofumi Nakano: Haruo Honda, all of Tokyo, Japan [73] Assignee: KyowaHakko Kogyo Co., Ltd., Tokyo, Japan [22] Filed: Apr. 3, 1973 [2]] Appl.No.: 347,399

' 30 Foreign Application Priority Data Apr, 5, 1972 Japan 47-33483 52us. Cl. 195/96, 195/80 R v [51] Int. Cl.. Cl2d 13/00 [58] Field ofSearch l95/96, 80 R, 112, 42, 195/79 [56] References Cited UNITED STATESPATENTS 2,483,892 10/1949 Ehrlich et al. 195/80 R 3,751,339 7 1973Suzuki eta]. l95/96 Primary Examiner-Lionel M. Shapiro AssistantE.\'amt'net-R0bert J. Warden Attorney, Agent, or FirmCr-aig & Antonelli[57] ABSTRACT 17 Claims, N0 Drawings 1 PROCESS or: PRODUCINGCHLOR'AMPHENICOL ANALOGS I Thisinvention relates to a process forproducing chloramphenicol analogs by fermentation. More particularly, itrelates to a process for producing chloramphenicol analogs, whichcomprises culturing a bacterium capable of producing chloramphenicolanalogs,

belonging to the genus Corynebacterium or Nocardia and monoanddiac'e'tyl derivatives thereof, exemplified by:

D-threo-2-isobuty ramide- 1 -p-riitrophenyl-1, 3-

propanediol (hereinafter referred to as compoundD-threo-Z-prOpionamide'l -'p-nitr'ophenyl-l, 3-

propanediol (hereinafter referred-to as compound II) I .H NHCOCH:

m Lawton D-threo-Z-acetamide-l-p-nitrophenyl-l, 3- propanediol-l,'3-diacety1ester (hereinafter referred to as compoundIV) n Nnooom Hcoon:

2 D-threo -2-propionamidel -p-nitropheny1- l 3-propanediol-3-acetylester(hereinafter referred to as compound V) H NHCO CHzCH;

(IRE;

D-threo-Z-acetamide- 1 -p-nitrophenyl-1,

3-propanediol-3-acetylester (hereinafter referred to as compound VI)D-threo-2-amino-l-p-nitrophenyl-1, 3-propanediol (hereinafter referredto as compound VII) Of these chloramphenicol analogs, the compounds I,II and III are utilized as antibiotics and the compounds IV to VI areutilized as raw materials for the preparation of the compounds 11 andIII. Further, the compounds I to VII are used as raw materials for thepreparation of chloramphenicol. The compounds IV, V and VI arehydrolyzed under mild conditions to result the compound II or III. Thecompounds 1 VI may be hydrolyzed to the compound VII, which is reactedwith dichloracetic' acidto produce-chloramphenicol.

As regards theproduction of chloramphenicol analogs of the compounds I,II and 111, there have been known processes which comprises theutilization of bacteria of the genus Arthrobacter, Corynebacterium orNocara'ia (U.S. Ser. No. 137,695 filed Apr. 26, 1971 riow U.S. "Pat. No.3,751,339 and No. 137,696 filed Apr. 26, 1971 now abandoned).

The present inventors, throughfurther studies, have found that abacterium belonging to the genus C0rynebacterium of Nocardia andha'vingaresistance to chloramphenicol or its analogs canproduce a considerableamount of chloramphenicol analogs of the compounds I VII, and haveestablished the present invention.

The present inventors have made studies in an attempt to further improvethe known bacteria whichare capable of producing chloramphenicol analogs(fo'r'example, the bacteria of the genera Corynebacterium and Nocardiacapable of producing chloramphenicol analogs, which are disclosed inU.S. Ser. No 137,695 filed Apr. 26, 1971, and No. 137,696 filed Apr. 26,1971).

As the result, ithas'been found that these bacteria have a considerablyhigh resistance to chloramphenicol as compared with enteric bacteria butare incapable of growing in the presence of about 15 40 ug/ml ofchlohaving'the ability to produce chloramphenicol analogs.

On the basis of these findings, the present inventors have obtainedmutant strains of bacteria belonging to the genus Corynebacterium orNocardia, which are capable of growing in the presence of a highconcentration of chloramphenicol or its analogs. Such mutant strains areresistant to generally more than about 40 tag/ml of chloramphenicolthough the maximum concentration of resistance varies depending upon thekinds of mutant strains. The present inventors have examined theproductivity of chloramphenicol analogs by these mutant strains andfound that they have a remarkably improved productivity ofchloramphenicol analogs as compared with the known strains.

In obtaining mutant strains having a resistance to chloramphenicol orits analogs used in the present invention, any of the conventionalmethods for inducing mutation to obtain a strain having a resistance maybe employed.

For example, a strain of the genus Corynebacterium or Nocardia, havingthe ability to produce chloramphenicol analogs (but having a very lowresistance to chloramphenicol or its analogs, for example,Corynebacterium hydrocarboclastus ATCC 21628) is selected and its cellsare treated according to the conventional artificial mutation means suchas X-ray irradiation, ultraviolet ray irradiation, nitrous acidtreatment, nitrogen-mustard treatment, nitrosoquanidine treatment, etc.After the treatment, the cells are cultured on an agar medium containingan appropriate amount of chloramphenicol or its analog (i.e., one of theD-threo-2-amino-l-phenyl-l, 3-propanediol derivatives, for example, theabove described compounds I to VII, D-threo-2-diehloroacetamidel-p-methylthiophenyl-l, 3-propanediol, etc.). Thus, a strain having aresistance to chloramphenicol or its analogs can be isolated. In thiscase, the resistibility to chloramphenicol possessed by the resultantmutant strain varies depending upon the concentration of chloramphenicolor its analog in the medium. In the present invention, usually amutantstrain having a resistance to 40 ug/ml or more of chloramphenicol iseffective.

More particularly, for example, a strain of bacterium capable ofproducing chloramphenicol analogs (Corynebacterium hydrocarboclastusATCC 21628) is preliminarily grown sufficiently in a nutrient mediumcomprising percent meat extract, 1 percent polypeptone, 0.5 percentyeast extract, 0.3 percent sodium chloride and 2 percent sucrose (pH7.3). This strain is sufficiently washed with an isotonic sodiumchloride solution and resuspended in the isotonic sodium chloridesolution in a concentration of about 5 X cells per 1 ml. 5 ml of thesuspension is placed in a Petri dish having a diameter of 9 cm andsubjected to ultraviolet ray irradiation from a distance of 30 cm aboveby a commercially available ultraviolet ray lamp of -W for 2 minutes,while occassionally shaking. The lethal ratio of the cells after theultraviolet ray irradiation is 99.99 percent. The cells are collected bycentrifuge and cultured in the above described nutrient media,respectively, further containing 200 ug/ml and 500 ag/ml ofchloramphenicol for 5 days. A portion of each of the cells thus grown isagain cultured to grow in the media having the same composition as aboveand similarly containing 200 p.g/ml and 500 pg/ml of chloramphenicolrespectively. The resultant culture liquors are diluted stepwise toappropriate concentrations and smeared onto solid media prepared byadding 2 percent agar to the abovedescribed chloramphenicolcontainingmedia.

The colonies grown on the solid media are transferred to solid mediahaving the same composition and the growth is examined. Thus, strainscapable of sufficiently growing in the presence of 200 #g/ml or 500ag/ml of chloramphenicol are obtained. Typical strains are namedCI-ICM-ll and CHCM-l56.

Table 1 shows the examples of the strains having a resistance tochloramphenicol obtained in this manner. As is apparent from the table,since the mutants can grow at a higher concentration of chloramphenicolthan the parent strains, the mutant strains have much enhancedresistance to chloramphenicol as compared with the parent strains.Although only the strains having a resistance to chloramphenicol areshown in the table, the strains having a resistance to the abovedescribed analogs of chloramphenicol also have a resistance tochloramphenicol. Therefore these strains are regarded to have a crossresistance and the mechanisms of the resistances are deemed to besimilar. Examples of culturing steps will be explained below. Any of thethus obtained mutant strains has a higher ability to producechloramphenicol analogs than the parent strains.

Table 1 Resistance to chloramphenicol Minimum inhibitory concen-Concentration of chloramphenicol in the medium employed for isolation (PS/ CHCM-ll (resistant strain) CHCM-l56 (resistant strain) Minimuminhibitory concentration of chloramphenicol (#g/ Norcardia paraffinicaATCC 21 I98 (parent strain) Concentration of chloramphenicol in themedium employed for isolation (#g/ NCCM-3 (resistant strain) NCCM-305(resistant strain) In carrying out the process of the present invention,n-paraffins or various hydrocarbon fractions containing n-paraffms are,for example, preferable as a carbon source. C to C and particularly C toC n-paraffins have been found to be effective. However, any hydrocarbonmay be employed so far as it can be utilized by the microorganisms.Carbohydrates such as fructose, glucose, sucrose, etc., sugar alcoholsuch as sorbitol, etc. alcohol, organic acids such as acetic acid, etc.may also be employed. As a nitrogen source, inorganic and organicnitrogen sources which are usually utilized may be employed. Infermentation, inorganic salts and growth factors necessary forpropagation which are used in the conventional fermentation are employedin addition to these main components.

More particularly, in carrying out the culturing, hydrocarbons orcarbohydrates may be used as main carbon sources. They are supplemented,for example, with organic nitrogen sources such as corn steep liquor,

yeast extract etc., inorganic metallic salts such as iron, manganese,magnesium, potassium, sodium etc., and growth-promoting factors toprovide a culture medium. The medium is sterilized and inoculated withthe microorganism. The culturing is carried out under aerobic conditionsat 45C. During the culturing, the pH is adjusted to 4-l0, preferably6-8, for example, by addition of urea solution, aqueous ammonia, ammoniaor ammonium carbonate solution. The culturing is generally completedafter 2 to 7 days.

Now, the examples of the present invention will be explained below butthese examples merely illustrate the present invention and do notrestrict the scope thereof.

EXAMPLE 1 Nocardia paraffinica NCCM-305 (PERM-P No. 1409) (ATCC 21785)is cultured with shaking for 24 hours in a medium comprising 1.0 percentmeat extract, 1.0 percent peptone, 0.5 percent sodium chloride and 2percent sorbitol and having a pH of 7.2 (before sterilization). Theresulting seed culture is inoculated at an inoculum ratio of 10 percent(V/V) in 3.0 l of a medium having the following composition which isplaced in a 5 [jar fermenter and sterilized;

KH PO, 0.2% NA HPO 0.2% MgSO -7H O 0.1% MnSO '4H O 0.002% FeSO.,.7l-1 O0.02% ZnSO '7H O 0.001% (NHmSO, 0.5% CuCI '2H O 0.0003% Corn steepliquor 0.5% Yeast extract 0.5%

n-Paraffins (a mixture of system of chloroform and methanol (93:7).Absorption spots are detected with ultraviolet rays. Each of the spotsis scraped out, extracted with a certain amount of methanol andsubjected to quantitative analysis by measuring an absorption at 278 mu.The compounds obtained are as follows:

Compound 1 Compound 11 Compound Ill At the same time, theabove-described procedure is carried out using the parent strain (ATCC21 198). The results of this run are as follows:

Compound I 0.5 g Compound 11 0.6 g/l Compound 111 0.2 g/l EXAMPLE 2Nocardia paraflinica NCCM-305 (PERM-P No. 1409) (ATCC 21,785) is used.Culturing is carried out for 'h0urs in the same manner as described inExample 1 except that 10 percent fructose is used in place ofn-paraftins. Fractionation and quantitative analysis are carried out inthe same manner as described in Example 1. As the result, the productionof the following compounds is revealed.

Compound I 1 Compound 11 0 Compound Ill 0 Compound IV 0. Compound V 0Compound V1 0 At the same time, the similar procedure is carried outusing the parent strain (ATCC 21198). The following compounds areobtained:

Compound 1 0.2 g/l Compound 11 0.15 g/l Compound 111 0.2 g/l Compound IVVl not detected EXAMPLE 3 Corynebacterium hydrocarboclastus CHCM-156(PERM-P No. 1408) ATCC 21,784) is used and culturing is carried out for80 hours in the same manner as described in Example 1. Fractionation andquantitative analysis are carried out in the same manner as described inExample 1. As the result, the production of the following compounds isrevealed:

Compound I 3 Compound 11 5 Compound Ill 4 Compound 1V 3 Compound V 1Compound VI 1 Compound VII 0 At the same time, the similar procedure iscarried out using the parent strain (ATCC 21628). The results of thisexperiment are as follows:

Compound 1 2.3 g/l Compound ll 4.0 g/l Compound 111 1.8 g/l Compound 1V0.3 g/l Compound V 0.3 g/l Compound V1 0.3 g/l Compound V11 not detectedEXAMPLE 4 Corynebacterium hydrocarboclastus CHCM-l 56 (PERM-P No. 1408)(ATCC 21784) is used and culturing is carried out for hours in the samemanner as described in Example 1 except that 10 percent sucrose is usedin place of n'paraffins.

Fractionation and quantitative analysis are carried out in the samemanner as described in Example 1. As the result, the production of thefollowing compounds is revealed.

Compound 1 2.7 g/l Compound 11 5.3 g/l Compound 111 4.2 g/l Compound IV3.1 g/l Compound V 1.0 g/l Compound V1 1.0 g/l Compound V11 0.2 g/l Thedistribution of the products is nearly the same as that of the productsin Example 3.

EXAMPLE 5 Nocardia paraffinica NCCM-3 (ATCC 21,924) is used. Culturingis carried out for 80 hours in the same manner as described inExample 1. Fractionation and quantitative analysis are carried out inthe same manner as described in Example 1. As the result, the productionof the following compounds is revealed.

Compound 1 1.0 g/l Compound 11 1.0 g/l Compound 111 0.5 g/l EXAMPLE 6Corynebacterium hydrocarboclastus V Cl-lCM-l 1 (ATCC 21,923) is used.Culturing is carried out for 85 hours in the same manner as described inExample 1. Fractionation and quantitative analysis are carried out inthe same manner as described in Example 1. As the result, the productionof the following compounds is revealed.

(PERM-P No. 1,408) (ATCC 21,784) and Nocardia paraffinica NCCM-305(FERM-P No. 1409) (ATCC 21,785) are used. Culturing is carried out for92 hours in the same manner as described in Example 1 except that 0.5percent (W/V) of sodium acetate is used in place of percent (V/V) ofn-paraffins. During culturing, a solution of 7 percent of ammoniumacetate in 50 percent acetic acid solution is added little by littlecontinuously to the medium as the microorganisms propagate. Total of 1.5l of the solution is added to the medium up to the completion ofculturing. After the completion of culturing, the following compoundsare produced in the culture liquor.

ATCC 21784 ATCC 21785 Compound 1 2.4 g/[ 1.0 g/l Compound 11 3.0 g/! 0.9g/l Compound 111 2.8 g/! 1.0 g/l Compound IV 0.8 g// 0.4 g/l Compound V0.7 g// 0.4 g/l Compound V1 0.4 g/l 0.4 g/l Compound Vll 0.2 g/l traccWhat we claim is:

1. A process for producing chloramphenicol analogs, which comprisesculturing a bacterium capable of producing chloramphenicol analogsbelonging to the genus Corynebacterium or Nocardia and having aresistance to chloramphenicol or its analogs in a nutrient medium andrecovering the chloramphenicol analogs from the resulting cultureliquor.

2. The process of claim 1, wherein the analogs are D-threo-2-amino-l-p-nitrophenyl-1, 3-propanediol, derivatives thereofwherein the 2-amino group is substituted by an acylamino group selectedfrom the group consisting of acetamide, propionamide, and isobutyramideor monoand di-acetyl derivatives thereof.

3. The process of claim 1, wherein the nutrient medium contains anassimilable carbon source and culturing is effected under aerobicconditions.

4. The process of claim 3, wherein said carbon source is a C to Cn-paraffin.

5. The process of claim 3, wherein said carbon source is a materialcontaining sucrose or fructose as sugar source.

6. The process of claim 3, wherein said carbon source is acetic acid.

7. The process of claim 1, wherein said bacterium is the genusCorynebacterium.

8. The process of claim 1, wherein said bacterium is the genus Nocardia.

9. The process of claim 1, wherein said bacterium is the speciesCorynebacterium hydrocarboclastus.

10. The process of claim 1, wherein said bacterium is the speciesNocardia paraffinica.

11. The process of claim 1, wherein said culturing occurs at 20C to 45Cat a pH of 4 to l0.

12. The process of claim 1, wherein the bacterium is a mutant strainwhich is capable of growing in the presence of a high concentration ofchloramphenicol or its analogs.

13. The process of claim 10, wherein the mutant strain of bacterium hasthe resistance to at least 40 ug/ml of chloramphenicol or its analogs.

14. The process of claim 1, wherein the bacterium is Corynebacteriumhydrocarboclastus ATCC 21,784.

15. The process of claim 1, wherein the bacterium is Corynebacteriumhydrocarboclastus ATCC 21,923.

16. The process of claim 1, wherein the bacterium is Nocara'iaparafflnica ATCC 21,785.

17. The process of claim 1, wherein the bacterium is Nocardiaparaffinica ATCC 21,924.

1. A PROCESS FOR PRODUCING CHLORAMPHENICOL ANALOGS, WHICH COMPRISESCULTURING A BACTERIUM CAPABLE OF PRODUCING CHLORAMPHENICOL ANALOGSBELONGING TO THE GENUS CORYNEBACTERIUM OR NOCARDIA AND HAVING ARESISTANCE TO CHLORAMPHENICOL OR ITS ANALOGS IN A NUTRIENT MEDIUM ANDRECOVERING THE CHLORAMPHENICOL ANAGOLS FROM THE RESULTING CULTURELIQUOR.
 2. The process of claim 1, wherein the analogs areD-threo-2-amino-1-p-nitrophenyl-1, 3-propanediol, derivatives thereofwherein the 2-amino group is substituted by an acylamino group selectedfrom the group consisting of acetamide, propionamide, and isobutyramideor mono- and di-acetyl derivatives thereof.
 3. The process of claim 1,wherein the nutrient medium contains an assimilable carbon source andculturing is effected under aerobic conditions.
 4. The process of claim3, wherein said carbon source is a C10 to C22 n-paraffin.
 5. The processof claim 3, wherein said carbon source is a material containing sucroseor fructose as sugar source.
 6. The process of claim 3, wherein saidcarbon source is acetic acid.
 7. The process of claim 1, wherein saidbacterium is the genus Corynebacterium.
 8. The process of claim 1,wherein said bacterium is the genus Nocardia.
 9. The process of claim 1,wherein said bacterium is the species Corynebacterium hydrocarboclastus.10. The process of claim 1, wherein said bacterium is the speciesNocardia paraffinica.
 11. The process of claim 1, wherein said culturingoccurs at 20*C to 45*C at a pH of 4 to
 10. 12. The process of claim 1,wherein the bacterium is a mutant strain which is capable of growing inthe presence of a high concentration of chloramphenicol or its analogs.13. The process of claim 10, wherein the mutant strain of bacterium hasthe resistance to at least 40 Mu g/ml of chloramphenicol or its analogs.14. The process of claim 1, wherein the bacterium is Corynebacteriumhydrocarboclastus ATCC 21,784.
 15. The process of claim 1, wherein thebacterium is Corynebacterium hydrocarboclastus ATCC 21,923.
 16. Theprocess of claim 1, wherein the bacterium is Nocardia paraffinica ATCC21,785.
 17. The process of claim 1, wherein the bacterium is Nocardiaparaffinica ATCC 21,924.